Peripheral T cell tolerance is considered to be an important immunological response that inhibits inappropriate immune responses to both self and non-self antigens (Ags). However, tolerance is breached in disease that can manifest as allergic or autoimmune disease. Using a murine model of tolerance, induced by inhaled Ag, we have recently made interesting discoveries. In tolerized mice, we have identified cells expressing membrane-bound TGF-beta and the T regulatory cell-specific transcription factor FOXP3. This novel cell type, TGF-betam+/FOXP3+, was not detectable in Ag-induced airway inflammation. Our studies have also allowed us to make an important distinction between soluble and membrane-bound TGF-beta. While soluble TGF-beta potently inhibited naive CD4+ T cell activation through inhibiting Ca2+ influx, Itk and NFAT activation, it was unable to inhibit Ag-experienced cells. The novel TGF-betam+/FOXP3+ - expressing cells, however, were very effective in inhibiting the effector functions of Ag-experienced cells. In our most recent studies, we have observed that TGF-betam+/FOXP3+ cells have the unique ability to upregulate Notch in target cells resulting in cleavage of Notch to the transcriptional activator form, Notch Intracellular Domain (NICD), which in turn induced HES1, known to be a transcriptional represser. Collectively, these studies lead us to hypothesize that a) membrane-bound TGF-beta plays an important role in Ag-induced tolerance induction in the respiratory tract and b) Co-operation between membrane-bound TGF-beta- and Notch/NICD-induced pathways is critical for induction of the tolerant state. To test this hypothesis we will: Aim I. Determine the role of membrane-bound TGF-beta and Notch as Common mediators utilized for tolerance induction in the respiratory tract. Studies will involve analysis of CD4+T cells from lung-draining lymph nodes (LNs) of mice subjected to two different models of tolerance and use of CD4-specific Notch knockout mice. Aim II. Determine functional distinctions between soluble and membrane-bound TGF-beta and the importance of Notch cleavage by membrane-bound TGF-beta as a critical step in tolerance induction. Molecular interactions between signaling molecules activated downstream of the TGF-beta receptor and Notch pathways in target cells will be investigated by biochemical, molecular and imaging techniques. Aim III. Characterize the effects of a TGF-beta-rich microenvironment on lung LN dendritic cells and their effects, in turn, on T cells and determine whether HES1 overexpression in a T cell-inducible fashion in mice results in inhibition of allergic airways disease. Expression of the Notch 1 ligand Jagged-1 will be studied on lung LN dendritic cells and the effect of these DCs on CD4+CD25- and CD4+CD25+ cells will be studied. Studies will also involve TGF-beta effects on lung and bone marrow-derived DCs. T cell-specific inducible HES1 transgenic mice will be generated to determine whether HES1 overexpression inhibits the development of allergic airways disease. Thus, using a variety of molecular, biochemical and immunological methods and genetically altered mice, our studies will focus on the co-operation between the TGF-beta and Notch pathways in tolerance induction in the respiratory tract.